Although it was previously demonstrated that T cell receptor-mediated interaction is necessary to thymic stromal development, the role of costimulatory signals in thymic development has not been assessed. Studies were therefore undertaken to determine whether costimulation influences cortico-medullary organogenesis and thymic repertoire selection. Mice with genetically altered expression of costimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40, or of the costimulatory receptors CD28 and CD154 (CD40 ligand) have been analyzed for thymic development and expressed T cell repertoire. Disruption of the CD28-B7 pathway or of the CD40-CD154 pathway had minimal effect on development of normal thymic cortico-medullary architecture. In contrast, disruption of both pathways resulted in a profound failure of thymic development. These findings identify a previously unappreciated role of redundant costimulatory pathways mediating an essential function in thymic development. The effect of these costimulatory pathways on selection of the T cell repertoire was studied in parallel. Two pathways of negative selection have been identified. One pathway is CD40L-dependent and acts at a relatively early stage in intra-thymic development and is mediated by a non-cell-autonomous mechanism. The second is CD40L-independent and occurs later in intra-thymic or post-thymic development. Interestingly, we have observed that when negative selection is abrogated by inactivation of the CD40L pathway, functional self tolerance is maintained by a non-deletional mechanism. This non-deletional tolerance is mediated by a CD28-dependent pathway. Thus, redundant pathways exist to protect against self reactivity in the thymus, and disruption of both C40L (deletional) and CD28 (non-deletional) mechanisms of self tolerance results in a population of highly self-reactive thymic T cells. Together, these findings have elucidated the roles of costimulatory pathways in thymic T cell repertoire selection and maintenance of self tolerance. We have also assessed the role of costimulatory pathways in peripheral mechanisms for tolerance. A model system of mouse pregnancy and tolerance to the male-specific H-Y antigen has been used to characterize both deletional and non-deletional mechanisms for peripheral tolerance. Both CD28/B7 and Fas/FasL interactions are necessary for deletional as well as non-deletional components of H-Y tolerance induction during pregnancy. The nature of host/fetal interactions in the tolerization process is being further analyzed.The expression of both CD28 and B7 is highly regulated during thymic development, such that the most immature thymocytes are protected from exposure to costimulatory signaling. We hypothesized that costimulation of immature thymocytes might disrupt normal differentiation, and addressed this hypothesis by genetically engineering ectopic expression of costimulatory receptor/ligand molecules. The transgenic over-expression of CD28 and CD86 had dramatic effects on thymic maturation, leading to differentiation of CD4+CD8+ thymocytes in the absence of TCR expression, suggesting a previously undescribed ability of costimulatory signaling to bypass pre-TCR function in T cell development. This observation has provided an opportunity to further define the requirements for pre-TCR in T cell development. Suppression of TCR beta chain rearrangement (beta suppression or allelic exclusion), regarded as a pre-TCR dependent function, is efficiently mediated by CD28 signaling in the absence of a pre-TCR. Ongoing studies are characterizing the mechanisms mediating these critical parameters of T cell development.